Risk factors for COVID-19 associated pulmonary aspergillosis and outcomes in patients with acute respiratory failure in a respiratory sub-intensive care unit

Background COVID-19-associated pulmonary aspergillosis (CAPA) is burdened by high mortality. Data are lacking about non-ICU patients. Aims of this study were to: (i) assess the incidence and prevalence of CAPA in a respiratory sub-intensive care unit, (ii) evaluate its risk factors and (iii) impact on in-hospital mortality. Secondary aims were to: (i) assess factors associated to mortality, and (ii) evaluate significant features in hematological patients. Materials and methods This was a single-center, retrospective study of COVID-19 patients with acute respiratory failure. A cohort of CAPA patients was compared to a non-CAPA cohort. Among patients with CAPA, a cohort of hematological patients was further compared to another of non-hematological patients. Results Three hundred fifty patients were included in the study. Median P/F ratio at the admission to sub-intensive unit was 225 mmHg (IQR 155–314). 55 (15.7%) developed CAPA (incidence of 5.5%). Eighteen had probable CAPA (37.3%), 37 (67.3%) possible CAPA and none proven CAPA. Diagnosis of CAPA occurred at a median of 17 days (IQR 12–31) from SARS-CoV-2 infection. Independent risk factors for CAPA were hematological malignancy [OR 1.74 (95%CI 0.75–4.37), p = 0.0003], lymphocytopenia [OR 2.29 (95%CI 1.12–4.86), p = 0.02], and COPD [OR 2.74 (95%CI 1.19–5.08), p = 0.014]. Mortality rate was higher in CAPA cohort (61.8% vs 22.7%, p < 0.0001). CAPA resulted an independent risk factor for in-hospital mortality [OR 2.92 (95%CI 1.47–5.89), p = 0.0024]. Among CAPA patients, age > 65 years resulted a predictor of mortality [OR 5.09 (95% CI 1.20–26.92), p = 0.035]. No differences were observed in hematological cohort. Conclusion CAPA is a life-threatening condition with high mortality rates. It should be promptly suspected, especially in case of hematological malignancy, COPD and lymphocytopenia. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-024-09283-3.

Nowadays, COVID-19-associated pulmonary aspergillosis (CAPA) has been recognized as a major complication of critically ill COVID-19 patients [4].According to ECMM/ISHAM, CAPA is classified into possible, probable, and proven [5].It is defined as IPA developing subsequent to SARS-CoV-2 infection and may be suspected in case of i) refractory fever for more than 3 days or new onset fever after a period of defervescence lasting longer than 48 h during appropriate antibiotic therapy, ii) worsening respiratory status despite receiving all recommended treatments for COVID-19; iii) hemoptysis; and iv) pleural friction rub or chest pain [5].
Diagnosis remains challenging due to the lack of strong consensus definitions and because clinical and radiological findings can mimic those of severe COVID-19 [5,[17][18][19].
Furthermore, data are lacking concerning non-ICU patients and possible clinical differences between hematological patients and non-hematological patients with CAPA.
Aims of this study were to (i) assess the incidence and prevalence of CAPA patients hospitalized in a respiratory sub-intensive care unit, (ii) evaluate the risk factors for CAPA development and (iii) examine the impact of CAPA on in-hospital mortality.Among the CAPA cohort, secondary aims were to (i) assess factors independently associated with mortality, (ii) evaluate clinical differences between hematological and non-hematological patients.

Study design
We conducted a single-center, retrospective study on patients with COVID-19 pneumonia and respiratory failure hospitalized in a respiratory sub-intensive care unit at Azienda Ospedaliero-Universitaria Policlinico Umberto I, Sapienza University of Rome, from January 2021 to December 2022.A cohort of CAPA patients was compared to a cohort of non-CAPA patients.Among patients with CAPA, a cohort of hematological patients was further compared to another of non-hematological ones.
Inclusion criteria were: (i) diagnosis of COVID-19 pneumonia and respiratory failure and/or acute respiratory distress syndrome (ARDS), (ii) hospitalization in the respiratory sub-intensive care unit for > 48 h and (iii) age > 18 years.Exclusion criteria included: age < 18 years, hospitalization in the respiratory sub-intensive care unit for < 48 h and missing data.
The study received approval from the local Ethics Committee (ID Prot.109/2020).

Setting
Starting from September 2020, we set up a respiratory sub-intensive care unit with 42 beds.
In our respiratory sub-intensive care unit patients were admitted in case of acute respiratory failure and/or ARDS due to COVID-19 pneumonia, requiring oxygen therapy and/or Helmet continuous positive airway pressure (CPAP) treatment or non-invasive mechanical ventilation (NIV).In patients with tracheostomy, invasive mechanical ventilation (IMV) was employed.
Patients required the use of continuous vital signs monitoring, and, in most cases, central venous catheter (CVC) or arterial catheters' placement, total parenteral nutrition and, in case of non-adaptation to ventilation, sedation.We administered dexmedetomidine for sedation, or morphine or midazolam as a secondary line in cases of inadequate response to dexmedetomidine.
Transfer to ICU was required if patients needed orotracheal intubation and IMV.

Clinical criteria and microbiological methods for diagnostic cultures and infection management
CAPA was defined according to recently proposed definitions [5] as well as practice guidelines [20] using a combination of clinical, radiological, and mycological features of the disease.
Respiratory samples included specimens such us tracheobronchial aspirate (TBA) and/or broncolavage (BAL) (when feasible) and were collected on clinical criteria.Bronchoscopy was not routinely performed and was deemed unfeasible, due to technical difficulties with performing an invasive exam in patients with severe respiratory failure who required CPAP and/or NIV.On respiratory samples, galactomannan (GM) and fungal culture were performed.Fungal cultures were incubated for 7 days at 30 °C on Sabouraud selective media, whereas GM test in serum, BAL and TBA was performed according to manufacturer's instructions (Platelia Aspergillus EIA, Bio-Rad).
In case of suspected CAPA, the clinical approach was managed together with a dedicated infectious disease specialist (author name, AO).When feasible, chest CT scan was repeated to detect lesions compatible with IPA and was analyzed by dedicated pneumologist and radiologist.In instances of uncertainty, a panel discussion was conducted.

Definitions
Respiratory failure was diagnosed for PaO 2 values < 60 mmHg at room air at arterial blood gases (ABGs) upon admission to our ward or to the emergency department, whereas PaO 2 / FiO 2 ratio (P/F ratio) was used as an indicator of severity, according to Berlin definitions [21].Only the P/F ratio at admission was included in the statistical analysis.Diagnosis of COVID-19 pneumonia relied on clinical data, ABGs and chest CT scan performed for all patients at hospital admission [22].Severe and critical disease were defined according to WHO definitions [23].
Prior (30-day) infections referred to infections diagnosed within 30 days before admission; prior (30-day) antibiotic exposure included receiving antibiotic therapy in the 30 days preceding the diagnosis of CAPA.Chronic steroid treatment was defined as the use of prednisone or its equivalent at a dosage of at least 0.5 mg/kg/day for a minimum of 30-days before admission.Immunodeficiency was defined as the presence of primitive or secondary immunodeficiency conditions (e.g., AIDS, active chemotherapy) [24].
APACHE II and Charlson Comorbidity Index (CCI) were collected as severity scores at admission.
All patients received a 10-day course of dexamethasone treatment at a dosage of 6 mg/daily due to COVID-19 severity (even patients initially presenting with moderate COVID-19 at admission worsened to a severe stage) and/or antiviral/monoclonal therapy according to available guidelines during the study period [26].Additionally, all patients received antithrombotic prophylaxis with enoxaparin.
Mortality referred to in-hospital death for all causes.

Statistical analysis
The data were presented as medians with interquartile ranges (IQR) for continuous variables and as simple frequencies, proportions, and percentages for categorical variables.Mann-Whitney test was used for unpaired samples.Dichotomous variables were compared using Fisher's exact tests or chi-square test statistics, as appropriate.Survival was analyzed via Kaplan-Meier curves and the statistical significance of differences between the two groups was assessed using the log-rank test.Multivariable logistic regression was conducted to identify independent predictors for CAPA development and for mortality.All statistical analyses were performed using Graph Pad Prism version 10.0.3.

General population
The study comprised 350 patients, with a median age of 73 years (IQR 62-83).Among them, 262 (74.8%) presented with severe or critical COVID-19 pneumonia with a median P/F ratio at admission of 225 mmHg (IQR 155-314).General characteristics of the study population are outlined in Table 1.
The general population was further divided into CAPA and non-CAPA cohorts (Table 1).Table 2 shows specific features of CAPA cohort.

CAPA cohort
As shown in Table 2, 55 (15.7%) patients developed CAPA, with an incidence of 5.5% over the two years of observation.
There were no cases of proven CAPA, 18 (32.7%)were classified as probable CAPA and 37 (67.3%) as possible CAPA [5].Mycological features of CAPA cohort are reported in Supplementary Table 1.
The diagnosis of probable/possible CAPA was established at a median of 17 days (IQR 12-31) from SARS-CoV-2 infection and at a median of 3 days (IQR 0-6) from the onset of CAPA symptoms.In 5 (9.1%) cases, the diagnosis occurred post-mortem.Mortality rate was 61.8% (Table 1).Death occurred at a median of 6 days (IQR 4-15) from CAPA diagnosis.At the time of CAPA diagnosis, most patients presented with severe respiratory failure with a median P/F ratio of 127 (IQR 88. 5-200.8).
Kaplan-Meier survival curves have demonstrated different mortality rates in patients with CAPA development and in those with lymphocytopenia (Fig. 1a-b).

Comparison between hematological and non-hematological patients with CAPA
Non-hematological patients displayed higher prevalence of cardiovascular disease, diabetes, and chronic kidney disease (p = 0.02, 0.03 and 0.005, respectively) (Table 5).Conversely, in line with their malignancy condition, they presented a higher percentage of immunodeficiency, prior 30-day chemotherapy and anti-CD20 therapy.Mortality rate was similar between the two cohorts, while the rate of transfer to ICU was higher in the hematological cohort [15% vs 8.6%, p = 0.66], albeit not statistically significant.
Kaplan-Meier curves at 30 days from admission did not differ in patients with hematological malignancy (Fig. 2).

Discussion
Our main findings were: i) in patients hospitalized in sub-intensive care unit for severe and/or critical COVID-19, CAPA represents a main complication and a risk factor for mortality even in immunocompetent patients; ii) CAPA is burdened by a high mortality rate, especially within the first days following diagnosis; iii) patients affected by hematological malignancy, COPD and lymphocytopenia are at heightened risk of developing CAPA.
To the best of our knowledge, this is the first study evaluating CAPA in non-ICU patients, reporting original data about a specific respiratory sub-intensive setting of care.Our findings confirm previous data from ICU studies [4,8].First, CAPA affected older patients with multiple comorbidities, who presented severe or critical COVID-19 and a higher APACHE II score at admission.Additionally, CAPA emerged as an independent risk factor for in-hospital mortality at multivariate analysis.Kaplan-Meier curves sustained this finding, showing a significant increase of mortality in CAPA patients at 30 days from admission.Remarkably, in most cases, patients died within a median of 6 days from diagnosis, underscoring the severity of the disease despite prompt antifungal therapy.Notably, 5 (9.1%) patients died during samples analysis time, meaning before a correct diagnosis and treatment could have been obtained.This data supports the urgency of early suspicion and diagnosis of CAPA in high-risk patients.
However, prompt diagnosis can be hindered by nonspecific clinical and radiological features of the disease [5].Indeed, in our cohort, main clinical manifestations included worsening of respiratory failure and fever.Likewise, we performed a chest CT scan only in 50.9% of patients, due to technical difficulties in transporting critically ill patients with rapid respiratory deterioration.Nevertheless, among those scanned, we observed, as main patterns of CAPA, lung infiltrates and nodules, confirming the nonspecific patterns already described in literature.
In this scenario, recognizing risk factors for CAPA is challenging.Previous studies proposed several risk factors [7,[12][13][14][15][16]27], such as older age, tocilizumab treatment and the need for mechanical ventilation.A recent study [4], confirmed tocilizumab as a risk factor for probable CAPA, together with COPD and prior 28-d steroid therapy.In our study tocilizumab treatment was more common in patients who developed CAPA (p = 0.02), especially if affected by hematological malignancy (p = 0.01), but it did not represent an independent predictor of CAPA.Likewise, the need for mechanical ventilation did not result as an independent risk factor for CAPA, although administered more commonly to patients who died, in line with the severity of their COVID-19 pneumonia related condition.
Hematological malignancies are traditionally considered a risk factor for IPA [28] and were more common in non-survivors in our study.Nevertheless, we did not observe significant differences in hematological cohort beyond factors related to hematological condition itself.In other terms, this could mean that mortality in CAPA cohort could be related only to CAPA and severe COVID-19.Indeed, Kaplan-Meier curves did not show a different probability of survival between hematological and non-hematological patients and at multivariate analysis, only age > 65 years was found to be an independent predictor of mortality in CAPA cohort.
COPD has been recently considered as an emerging risk factor for IPA, in patients on chronic corticosteroid therapy [29].Chronic steroid treatment was more common in CAPA cohort but it did not predict CAPA development at multivariate analysis.Regarding the use of dexamethasone, recent evidence suggested that it increases the risk of CAPA [30,31], so its administration could have represented a possible risk factor in our cohort.Nevertheless, we did not investigate the role of dexamethasone since all patients received this Fig. 2 Kaplan-Meier curves in CAPA cohort.Kaplan-Meier curves showing survival at 30-d from the admission according to the presence of hematological malignancy in CAPA cohort treatment.Interestingly, our cohort included COPD patients at any stage of the disease and with any treatment regimen suggesting that COPD itself can be a risk factor for CAPA regardless of chronic steroid treatment, in line with previous studies [15,16].
An important finding of our study is the association of lymphocytopenia with poor prognosis.Several authors already reported lymphocytopenia as a risk factor for CAPA [32,33] and as a predictor of severity in COVID-19 patients [9].In our study, lymphocytopenia is a predictor of poor prognosis and correlate both with mortality in overall COVID-19 population and CAPA development.Kaplan-Meier curves at 30-days showed a significant increase in mortality in patients with lymphocytopenia.As a matter of interest, in the CAPA population, no differences in terms of lymphocytopenia are observed between survivors and non-survivors.This suggests that in most patients lymphocytopenia could be caused by SARS-CoV-2 infection [30,34].Consequently, CAPA could develop also in immunocompetent patients who experience a transient immunocompromise condition [32].Indeed, severe COVID-19 is known to decrease the number and functionality of CD4 + T and CD8 + T-cells and induce a hyperinflammatory state that enhances fungal growth [32][33][34].Lymphocytopenia has also been already identified as a predictor of influenza associated pulmonary aspergillosis (IAPA) [35].Moreover, data from lung transplant recipients reported a high mortality in patients affected by respiratory viral illnesses and IPA superinfection [31].These evidences suggest that a possible relationship between other respiratory viruses and secondary fungal infections should be considered and further investigated.
Our study has some limitations.It is a single-center, retrospective study.Patients in both cohorts were admitted in different pandemic periods.Variations in the pathogenicity of different SARS-CoV-2 variants and the improvement in medical staff experience and treatment efficacy over time, may have influenced patient outcomes.Presented data reflect a real-life scenario with no predefined CAPA screening protocol and diagnosis was mainly based on clinical suspicion.Moreover, bronchoscopy was not routinely performed and, accordingly, we mainly diagnosed possible rather than probable CAPA.We acknowledge that using the ECMM/ISHAM diagnostic criteria may be limited by the fact that our patients were not hospitalized in the ICU at the moment of CAPA.However, since specific guidelines for CAPA in sub-intensive care units are lacking, we were forced to rely on guidelines that apply to the setting most similar to ours, namely the ICU setting.Finally, not all patients repeated chest CT scan at clinical worsening.
In conclusion, CAPA is a life-threatening condition in patients hospitalized in respiratory sub-intensive care unit for severe COVID-19, even among immunocompetent patients.Given its high short-term mortality rate, CAPA should be promptly suspected in patients experiencing respiratory worsening despite appropriate COVID-19 treatment, especially in those affected by hematological malignancies, COPD and lymphocytopenia.

Fig. 1 a
Fig. 1 a-b.Kaplan-Meier survival curves in overall population.a Kaplan-Meier survival curves showing differences in survival at 30-d in CAPA cohort versus non-CAPA cohort; (b) Kaplan-Meier survival curves showing differences in survival at 30-d according to lymphocytopenia

Table 1 ,
patients with CAPA presented a high rate of severe or critical COVID-19 at admission

Table 1
Baseline characteristics of patientsCOPD chronic obstructive pulmonary disease, BMI body mass index, CCI Charlson Comorbidity Index, paO 2 arterial oxygen tension, FiO 2 fraction of inspired oxygen, ICU intensive care unit, IMV invasive mechanical ventilation, HFNC high flow nasal cannula, CPAP continuous positive airways pressure, NIV non-invasive mechanical ventilation a Lymphocytopenia is defined as lymphocytes count inferior to 750 cells × 10 3 /mm3 [23]ver or critical COVID-19 was defined according to WHO definitions[23]

Table 2
Characteristics of CAPA cohort PaO 2 arterial oxygen tension, FiO 2 fraction of inspired oxygen, HFNC high flow nasal cannula, CPAP continuous positive airways pressure, NIV non-invasive mechanical ventilation a Chest CT was performed only in 28 patients b 2 BAL, 53 TBA c 6 samples d In 5 cases CAPA was diagnosed post-mortem so patients did not received any treatment CAPA cohort (n = 55)

Table 3
[23]ivariate analyses evaluating risk factors for CAPA development (panel A) and mortality in the overall population (panel B) and in the CAPA cohort (panel C)COPD chronic obstructive pulmonary disease, APACHE II acute physiology and chronic health evaluation II, CCI Charlson Comorbidity Index a Lymphocytopenia is defined as lymphocytes count inferior to 750 cells × 10 3 /mm 3 b Sever or critical COVID-19 was defined according to WHO definitions[23]

Table 4
Comparison between survivors and non-survivors in general population and in CAPA cohort

Table 4
[23]tinued)COPD chronic obstructive pulmonary disease, BMI body mass index, CCI Charlson Comorbidity Index, paO 2 arterial oxygen tension, FiO 2 fraction of inspired oxygen, ICU intensive care unit, IMV invasive mechanical ventilation, HFNC high flow nasal cannula, CPAP continuous positive airways pressure, NIV non-invasive mechanical ventilation a Lymphocytopenia is defined as lymphocytes count inferior to 750 cells × 10 3 /mm 3 b Sever or critical COVID-19 was defined according to WHO definitions[23]

Table 5
Comparison between hematological and non-hematological patients with CAPACOPD chronic obstructive pulmonary disease, BMI body mass index, CCI Charlson Comorbity Index, paO 2 arterial oxygen tension, FiO 2 fraction of inspired oxygen, ICU intensive care unit, IMV invasive mechanical ventilation, HFNC high flow nasal cannula, CPAP continuous positive airways pressure, NIV non-invasive mechanical ventilation a Lymphocytopenia is defined as lymphocytes count inferior to 750 cells × 10 3 /mm 3 b Sever or critical COVID-19 was defined according to WHO definitions[76]